DESCRIPTION (provided by applicant: RNA silencing pathways operate in all eukaryotic kingdoms and have been implicated in a range of processes including translational silencing, post-transcriptional RNA degradation, transcriptional gene silencing due to heterochromatin formation or DNA methylation and meiotic elimination or silencing of unpaired DNA. RNA silencing has also been shown to serve as an antiviral defense mechanism in plants, invertebrates, and fungi and to modulate animal virus infections. The antiviral function of RNA silencing not only protects an organism against virus infection, but the generation of virus-derived small RNA products (vsRNAs) and the expression of counter-acting virus-encoded suppressors of RNA silencing can also significantly influence host gene expression and phenotype by impinging on the larger RNA silencing pathway. Although considerable progress has been made in elucidating the core features of the RNA silencing antiviral defense response, and essential components, both host- and virus-encoded, involved in this interaction have been identified, many important details remain unresolved. The Specific Aims of this proposal are designed to address outstanding questions related to a) the origins and properties of vsRNAs, b) the potential for these small RNAs to alter host gene expression in persistent virus infections, and c) molecular mechanisms involved in virus-mediated induction and suppression of RNA silencing pathways. The Research Design takes advantage of a powerful experimental system consisting of a tractable eukaryotic organism, the chestnut blight fungus Cryphonectria parasitica, with a simplified antiviral pathway (involving a single Dicer and single Argonaute) that is the natural host of a constellation of well-characterized viruses with different replication strategies and properties representing five different virus taxonomic families. The results derived from completion of the proposed research will have broad relevance to ongoing efforts to understand the complex interactions between viruses and cellular RNA silencing pathways and the impact that these interactions have on the host. The influence of fungal RNA silencing antiviral pathways clearly needs to be considered in any current and future use of mycoviruses for experimental or therapeutic manipulation of the fungal host, e.g., biological control of fungal virulence or mycotoxin production. Finally, preliminary data already indicates that the evolutionary position of fungi relative to animals and plants will provide insights into novel mechanisms for the induction and suppression of RNA silencing pathways yet to be revealed in the other organisms. PUBLIC HEALTH RELEVANCE: The interactions between mycovirus infections and highly conserved antiviral RNA silencing defense pathways have the potential to influence gene expression, virulence and evolution of both medically relevant and agriculturally important fungal species. The proposed research takes advantage of a highly tractable experimental system to address unresolved questions with general relevance to eukaryotic virus/host interactions concerning the biogenesis of virus-derived small RNAs as a result of RNA silencing-based antiviral defense, the potential for these small RNAs to alter host gene expression in persistent virus infections, and molecular mechanisms involved in virus-mediated induction and suppression of RNA silencing.